Journal: Cell death & disease

Article Title: LL37 complexed to double-stranded RNA induces RIG-I-like receptor signalling and Gasdermin E activation facilitating IL-36γ release from keratinocytes.

doi: 10.1038/s41419-025-07537-9

Figure Lengend Snippet: Fig. 3 LL37/Poly(I:C) complexes induce IL-36γ release from keratinocytes independent of inflammasome and GSDMD activity. A HPKs were primed with IL-17A (100 ng/ml) or left unprimed (medium control) for 6 h prior to stimulation with Poly(I:C), Poly(dA:dT), LL37/Poly(I:C) or LL37/Poly(dA:dT) complexes (all 5 μg/ml) for 18 h. Supernatants were analysed for IL-36γ release by ELISA. B, C GSDMD-deficient N/TERT-1 cells (sgGSDMD) were stimulated with IL-17A (100 ng/ml) for 6 h prior to stimulation with LL37/Poly(I:C) complexes (5 μg/ml), VbP (1 μM), or transfected with Poly(I:C) (1 μg/ml) for 18 h. Supernatants were analysed for D IL-36γ or E IL-1β levels by ELISA. D, E HPKs were stimulated with IL-17A (10 ng/ml) and TNF-α (5 ng/ml) for 5 h, followed by inhibitor treatment with SB 203580 (p38i, 20 μM), Belnacasan (Casp1i, 10 μM), or DMSO for 1 h before stimulation with LL37/Poly(I:C) complexes (5 μg/ml), VbP (1 μM), or anisomycin (ANS, 1 μM) for 18 h. Supernatants were measured for D IL-36γ or E IL-1β by ELISA. F HPKs were primed with IL-17A for 5 h, followed by inhibitor treatment with MCC950 or DMSO for 1 h before stimulation with LL37/Poly(I:C) complexes for 18 h. Cells were treated with MCC950 1 h before stimulation. Supernatants were measured for IL-36γ release by ELISA. Data are presented (A–F) as the mean ±S.E.M. of 3 independent experiments and analysed with (A–C, F) two-way ANOVA and Šidák’s or (D, E) two-way ANOVA followed by Dunnett’s multiple comparisons test. *p < 0.05, **p < 0.01, ****p < 0.0001. ns non-significant, Lipo Lipofectamine 2000, M protein marker, sg single-guide RNA.

Article Snippet: HPKs were transfected by the 1 μg/ml HMW Poly(I:C) coupled with biotin (Invivogen) using Lipofectamine 2000, or treated with 1 μg/ml LL37 with and without pre-incubation with 1 μg/ml HMW Poly(I:C) coupled with Biotin (Invivogen).

Techniques: Activity Assay, Control, Enzyme-linked Immunosorbent Assay, Transfection, Marker

Journal: Cell death & disease

Article Title: LL37 complexed to double-stranded RNA induces RIG-I-like receptor signalling and Gasdermin E activation facilitating IL-36γ release from keratinocytes.

doi: 10.1038/s41419-025-07537-9

Figure Lengend Snippet: Fig. 4 LL37/Poly(I:C) complexes activate Caspase-3 and GSDME cleavage in human primary keratinocytes. A, B HPKs were stimulated with LL37/Poly(I:C) complexes (5 μg/ml) or were transfected with Poly(I:C) (1 μg/ml) for indicated times control treatment (−) indicates Medium or Lipo, respectively. Supernatants and cell lysates were subjected to SDS-PAGE and immunoblotting with indicated antibodies. * indicates non- specific band (C, D) GSDME-deficient N/TERT-1 cells (sgGSDME) were (D) primed with IL-17A (100 ng/ml) or (C) left unprimed for 6 h prior to transfection with Lipo/Poly(I:C) (1 μg/ml), stimulation with LL37/Poly(I:C) complexes (5 μg/ml) or one UVB pulse (0.0875 J/cm2) for 18 h. Data are presented as a representative (A, B) of three independent experiments or are presented (C, D) as the mean ±S.E.M. of 3 independent experiments and analysed with two-way ANOVA and Šidák’s multiple comparisons test. **p < 0.01, ***p < 0.001, ****p < 0.0001. ns non- significant, Lipo Lipofectamine 2000, M protein marker, sg single-guide RNA.

Article Snippet: HPKs were transfected by the 1 μg/ml HMW Poly(I:C) coupled with biotin (Invivogen) using Lipofectamine 2000, or treated with 1 μg/ml LL37 with and without pre-incubation with 1 μg/ml HMW Poly(I:C) coupled with Biotin (Invivogen).

Techniques: Transfection, Control, SDS Page, Western Blot, Marker

Journal: mSphere

Article Title: Host Cathelicidin Exacerbates Group B Streptococcus Urinary Tract Infection

doi: 10.1128/mSphere.00932-19

Figure Lengend Snippet: Susceptibility to cathelicidin is similar between UPEC and GBS clinical isolates. (A and B) MIC assays for COH1 and CFT073 using human LL-37 or murine CRAMP) in RPMI 1640 medium. (C) Killing kinetics of three GBS strains by 36 μg/ml (8 μM) LL-37 in RPMI 1640 medium over time. (D and E) MIC assays for COH1 and CFT073 using human LL-37 or murine CRAMP in synthetic urine. Symbols represent the means of independent experimental replicates ( n = 3 to 5/group), with lines indicating means and SEM. (F) MIC values of GBS clinical isolates collected from urine, vagina, skin, or blood. Symbols represent the means of three independent experimental replicates ( n = 6 to 16/group). Data were analyzed by Kruskal-Wallis with Dunn’s multiple-comparison test. RFU, relative fluorescence units.

Article Snippet: Mouse anti-LL-37 (HM2071; Hycult) antibody or rabbit anti-CRAMP (pAb-antiCAMP/LL-37, NB100-98689; Novus) antibody was added at a 1:2,000 dilution and incubated, with rocking, for 2 h at room temperature.

Techniques: Fluorescence

Journal: mSphere

Article Title: Host Cathelicidin Exacerbates Group B Streptococcus Urinary Tract Infection

doi: 10.1128/mSphere.00932-19

Figure Lengend Snippet: GBS infection limits bladder epithelium cathelicidin production, and GBS proteases degrade cathelicidin. (A and B) Human bladder epithelial cells (HTB-9) were infected with GBS A909, COH1, or NCTC 10/84 or with GBS COH1 and UPEC CFT073, as indicated, for 4 h at an MOI of 10. LL-37 production was measured by ELISA. (C) HTB-9 cells were infected with GBS COH1 for 3 h at an MOI of 10 or 1. LL-37 mRNA transcripts were quantified by qPCR and normalized to the level of the housekeeping gene, GAPDH, using the ΔΔ C T method. Symbols represent the means of independent experimental replicates ( n = 3 to 6/group), with lines indicating medians and interquartile ranges. (D) GBS strains A909, COH1, and NCTC 10/84 were incubated with 9 μg/ml (2 μM) LL-37 for 4 h with or without addition of protease inhibitors (PI). Samples were spotted onto a nitrocellulose membrane and probed for LL-37. Densitometry was normalized to the signal from the 9-μg/ml LL-37 input. Raw images of dot blots are depicted in Fig. S1 in the supplemental material. (E) Susceptibility of GBS COH1 to 27 μg/ml (6 μM) LL-37 with or without protease inhibitors (PI) was measured over 4 h by serial dilution and plating. Symbols represent the means of three independent experiments, and lines indicate the mean values of experimental replicates. (F) MIC assay of GBS COH1 using human LL-37 in RPMI 1640 medium with (white bars) or without (gray bars) addition of protease inhibitors. Symbols represent the means of independent experimental replicates ( n = 5/group), with lines indicating means and SEM. (G) Human bladder epithelial cells (HTB-9) were infected with GBS COH1 and UPEC CFT073 for 4 h at an MOI of 10 with or without addition of protease inhibitors. LL-37 production was measured by ELISA. Symbols represent the means of independent experimental replicates ( n = 6 to 8/group), with lines indicating medians and interquartile ranges. Data were analyzed by Friedman’s test with Dunn’s multiple-comparison test (panels A to C), two-way repeated-measures ANOVA with Dunnett’s multiple-comparison test (panel E), or two-way ANOVA with Sidak’s multiple-comparison test (panels D and G). *** * , P < 0.0001; * * , P < 0.01; * , P < 0.05.

Article Snippet: Mouse anti-LL-37 (HM2071; Hycult) antibody or rabbit anti-CRAMP (pAb-antiCAMP/LL-37, NB100-98689; Novus) antibody was added at a 1:2,000 dilution and incubated, with rocking, for 2 h at room temperature.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Incubation, Serial Dilution